【作者】 刘群；

【导师】 李名扬；

【作者基本信息】 西南大学 ， 园艺学， 2009， 博士

【摘要】 植物非特异性脂转移蛋白(nonspecific lipid transfer protein,nsLTP)是一类广泛存在于高等植物中的碱性小分子蛋白,编码nsLTP的基因在许多植物中都以基因家族的形式存在,如拟南芥、大麦等。nsLTP成员具有不同的生物学功能,主要涉及角质合成,用以调节脂质储藏的分解代谢的β氧化、体细胞胚胎形成、植物信号转导、参与植物有性生殖、花粉发育、对病原物的防御以及对生物和非生物胁迫的应答等。明确不同成员在植物抗性形成方面的作用,对于调控植物抗性和利用基因工程改良植物抗性有重要意义。本论文克隆得到了蜡梅脂转移基因家族4个成员,并进行了启动子分离工作,利用生物信息学、荧光定量PCR、原核表达等方法对获得的蜡梅脂转移蛋白基因家族成员抗逆性的差异进行了分析,主要结果如下:1蜡梅nsLTP基因家族4个成员的克隆与分子特征分析在已构建的蜡梅花cDNA文库及EST分析的基础上,通过对文库cDNA克隆全长测序,得到了4个编码蜡梅非特异性脂转移蛋白的基因(nsLTP),分别命名为CpLTP1,CpLTP2,CpLTP3和CpLTP4,GenBank登录号为FJ889521,FJ904082,FJ904083和FJ904084。它们的cDNA全长分别为611、1016、656和997 bp,ORF分别为360、360、351和660 bp,无内含子,5′和3′端的非翻译区的长度不等,存在的调控基序有所不同。运用生物信息学手段对蜡梅nsLTP基因家族4个成员编码蛋白CpLTP1、CpLTP2、CpLTP3和CpLTP4的结构特点与性质进行了分析,CpLTP1、CpLTP2、CpLTP3具有明显的植物脂转移蛋白nsLTPI类的特征,分子量都为9KD,含有保守的4个螺旋区、8个半胱氨酸位点和脂质结合基序,具有明显的信号肽序列,定位于细胞外的可能性最高,进一步的三级结构预测也显示能够形成管状的疏水腔。而CpLTP4与其他3个编码蛋白显著不同,具有较高的分子量(19.7KD),虽然同样具有8个保守的半胱氨酸位点,但第8个位点的位置与nsLTPI类有一定的差异,细胞定位有所不同,定位于质膜的可能性最高,三级结构预测显示形成上宽下窄的类似于三角形的疏水腔,聚类分析时与同样高分子量的刚毛怪柳、蓖麻等的脂转移蛋白聚在一支,这种高分子量的脂转移蛋白有可能形成一个新的类别。2蜡梅nsLTP基因家族4个成员的原核表达及其产物抑菌活性分析成功构建LTP1-pET、LTP2-pET、LTP3-pET、LTP4-pET原核表达载体,转化Origami(DE3)表达菌株,通过诱导条件优化,在28℃、0.5m MIPTG、诱导6h能够获得较好的可溶性表达。利用His-Bind蛋白纯化回收试剂盒获得4个纯化的重组蛋白。体外抑菌试验结果表明,4个重组蛋白都具有抑菌活性但有差异,其中LTP4重组蛋白抑制小麦赤霉菌生长的能力最强,LTP1最弱。对于细菌的抑制活性还有待进一步试验验证。3蜡梅nsLTP基因家族4个成员非生物胁迫表达的荧光定量分析对蜡梅植株进行低温、干旱、高盐和ABA诱导处理,利用荧光定量PCR技术分析蜡梅nsLTP基因家族4个成员在蜡梅叶片中的表达情况,结果表明,4个nsLTP基因在蜡梅幼苗中的表达受干早、ABA、低温和NaCl处理的影响程度不同。CpLTP1、CpLTP2、CpLTP3基因在胁迫处理下表达总体下调,而CpLTP4基因在胁迫处理下表达总体上调,其中在低温处理下表达量最高。结果表明虽然CpLTP1、CpLTP2、CpLTP3基因来自于同一基因家族,但其功能上具有明显的差异,可能在蜡梅幼苗的水分平衡、耐受低温、离子代谢等过程中发挥着不同的作用,CpLTP4基因可能对蜡梅抵抗非生物胁迫起更重要的作用。4蜡梅CpLTP3、CpLTP4基因启动子的克隆及瞬时表达分析利用hiTAIL-PCR法分离分别得到了CpLTP3、CpLTP4基因5′端上游的一段长1298bp和838bp的序列,二者的序列差异较大,Identity值为27.75%。经序列测定及软件分析表明,这两个序列具有典型的启动子结构,除含有TATA-box、CAAT-box等启动子基本元件,另外都含有较多的光应答元件、与植物非生物胁迫相关的一些响应元件,如ABRE、G-BOX、HSE。仅在CpLTP4pro发现GARE-motif、TATC-box、MBS、TC-rich repeats,在CpLTP3pro启动子发现与细胞周期调控有关的顺式调控元件MSA-like和胚乳表达必须的顺式调控元件Skn-1_motif。将克隆得到的蜡梅启动子CpLTP3pro、CpLTP4pro代替pBI121中的CaMV35S启动子构建成适于瞬时表达研究的植物表达载体pB1121-CpLTP3pro和pBI121-CpLTP4pro。通过农杆菌介导的瞬时表达研究,在GA3诱导下,CpLTP4pro能够驱动GUS基因在烟草叶片中的表达,而CpLTP3pro不能。在CpLTP3pro、CpLTP4pro驱动下,GUS基因在烟草叶片中的表达都受ABA、Nacl、PEG、4℃、37℃等处理诱导。表明蜡梅启动子CpLTP3pro、CpLTP4pro具有作为胁迫诱导驱动目标基因表达的潜力。论文研究结果表明,蜡梅nsLTP基因家族4个成员CpLTP1,CpLTP2,CpLTP3和CpLTP4的抗逆性存在差异,CpLTP4可能与蜡梅抗逆性的关系最为密切。为了进一步鉴别蜡梅nsLTP基因家族成员的抗逆性差异,尚需克隆蜡梅nsLTP基因家族其他成员及启动子,并利用转基因等技术对编码蛋白的抗逆性和启动子活性的诱导差异性进行深入研究才能完成。更多还原

【Abstract】 Nonspecific lipid transfer protein(nsLTP) is one kind of the low molecular weight basic proteins, which is widespread in higher plants.The genes encoding the nsLTP are usually in the form of gene family in many plants,such as arabidopsis,barley,et al.The nsLTP memebers have the different biological functions mainly involving synthesis of cutin,regulation of theβ-oxidation in the lipid storage catabolism,formation of somatic embryo,plant signal conduction,plant sexual reproduction,pollen development,defense against the pathogen and the response to the biotic and abiotic stresses,et al.In this paper,four genes encoding the nonspecific lipid transfer protein and two promoters were cloned from Chimonanthus praecox(L.) Link,and the bioinformatics,real time quantitative PCR, prokaryotic expression were used to analyze the difference of the stress resistance among the four Chimonanthus praecox nonspecific lipid transfer protein gene family members.The main results are as follows:(1) Cloning and molecular characteristics of four nsLTP genes from Chimonanthus praecoxWe cloned four nsLTP genes,named CpLTP1,CpLTP2,CpLTP3 and CpLTP4,the Genebank accession numbers of which are FJ889521,FJ904082,FJ904083and FJ904084 respectively,based on the cDNA library construction from Chimonanthus Praecox flower and its EST analysis.The full-length cDNA of the four genes are 611,1016,656 and 997 bp respectively,with similar opening reading frame(ORF) of 360 bp,360 bp,351 and 660 bp without the intron,but have different 5’ and 3’ untranslated region length and different regulation motifs.Analysis of the structure characteristics and properties of the four nsLTP genes by bioinformatics: With similar molecular weight of 9KD,the CpLTP1,CpLTP2 and CpLTP3 have the significant features of the plant nonspecific lipid transfer protein I(nsLTPI),containing four conversedα-Helix,eight cysteine residues and lipid-binding motifs,obvious signal peptide sequence.All the three proteins are most probably localized in extracellular,and can form the tubular hydrophobic cavity by further tertiary structure prediction.On the contrary,CpLTP4 is very different from the other three encoding proteins.It has the high molecular weight(19.7KD).Although CpLTP4 also has the eight conversed cysteine residues,the position of the eighth cysteine residues exhibits some differences from the nsLTPI type. The CpLTP4 is most likely to be located in the plasma membrane,which is different from the other three members.The tertiary structure prediction indicates that it can form the wide top and narrow bottom triangle-shaped hydrophobic cavity.Cluster Analysis shows that it can get together with the tamarix hispida and ricinus communis,maybe they can form a new type of the lipid transfer protein.(2) Prokaryotic expression and analysis of antibacterial activity against product of four nsLTP genes from Chimonanthus praecox in Escherichia ColiThe Prokaryotic expression vector LTP1-pET,LTP2-pET,LTP3-pET and LTP4-pET were constructed and transformed into the expression strain Origami(DE3).A fairly good soluble expression can be gained under the conditions of 28℃,0.5m MIPTG and 6h induced by optimizing the induced conditions.Four purified recombinant protein were obtained by the His-Bind protein Purification Kit.The results of the bacteriostatic test in vitro sbow that both of the four recombinant protein have the different antibacterial activity.The LTP4-pET recombinant protein has the most strong ability to inhibit the wheat phytoalexin where as the LTP1-pET recombinant protein has the weakest ability on it.The antibacterial activity of the four recombinant proteins against the bacteria should be verified by further research.(3) Abiotic stresses respond analysis of four nsLTP genes from Chimonanthus praecox by real time quantitative PCR.The Chimonanthus praecox plants were treated by cold temperature,drought,high salt and ABA. Then real-time quantitative RT-PCR was used to analyse the expression of four nsLTP genes in Chimonanthus praecox leaves.The results revealed that the four genes were differently regulated by drought,salinity,cold and ABA(abscisic acid).The expression of CpLTP1,CpLTP2 and CpLTP3 genes were general down-regulated by the stress treatment.But the expression of CpLTP4 was general up-regulated and had the highest expression under the cold treatment.The results suggest that although CpLTP1,CpL TP2 and CpLTP3 genes are from the same gene family,they have the distinct differences in functions and probably play different roles in water balance,chilling tolerance, metabolism and other physiological processes of Chimonanthus Praecox.CpLTP4 may play a more important role in resisting the abiotic stress of Chimonanthus Praecox.(4) Isolation and transient expression assays analysis of Chimonanthus praecox CpLTP3 and CpLTP4 promoterThe promoter fragment of 1298bp and 838bp upstream from the 5’ upper of the CpLTP3 and CpLTP4 genes was isolated from the genomic DNA of Chimonanthus praecox respectively by hiTAIL-PCR.There is great difference between the two sequences with the identity value of 27.75%. The sequencing and soft analysis suggest that the two sequences have the typical promoter structure. Besides the basic promoter elements like TATA-box,CAAT-box and so on,both of the CpLTP3 and CpLTP4 genes contain many light responsive elements,cis-acting element involved in the plant abiotic stress such as ABRE,G-box and HSE.Some elements such as GARE-motif,TATC-b0x,MBS and TC-rich repeats were only found in the CpLTP4pro sequence,while the cis-acting element MSA-like involved in cell cycle regulation,and cis-acting regulatory element Skn-1_motif required for endosperm expression were found in CpLTP3pro promoter sequence.In order to research the transient expression assays,plant expression vector pBI121- CpLTP3pro and pBI121- CpLTP4pro were constructed by replacing the CaMV35S promoter in pBI121 vector by the Chimonanthus praecox CpLTP3 and CpLTP4 promoterUsing leaf disc transformation method,pBI121-CpLTP3pro and pBI121-CpLTP4pro were transfered into tobacco(W38).The results show that under the induction of GA3,CpLTP4pro promoter can drive the expression of GUS gene,but the CpLTP3pro promoter doesn’t have the same ability.In Agrobacterium-mediated transient expression assay,activation of the CpLTP3 and CpLTP4 promoter region(1298bp and 838bp long) occurs in tobacco leaves after treatments with ABA,Nacl,PEG,cold temperature(4℃) and high temperature(37℃) which indicates that the Chimonanthus praecox CpLTP3 and CpLTP4 promoter have the potential of driving the expression of the target genes under the induced condition.To sum up the above results,four nsLTP Genes CpLTP1,CpLTP2,CpLTP3 and CpLTP4 from Chimonanthus praecox(L.) Link have the different stress resistance,CpLTP4 gene probably has the closest relationship with the stress resistance of Chimonanthus praecox(L.) Link.In order to identify the difference among the Chimonanthus praecox(L.) Link nsLTP gene family members,the other gene members and their promoters should be cloned.It is necessary to do some deep research on exploring the differences of the encoding proteins’ stress resistance and promoter inducing activity by transgenic and other related technologies.更多还原